A critical role for ecdysone response genes in regulating egg production in adult female Rhodnius prolixus

Ecdysteroids control ovary growth and egg production through a complex gene hierarchy. In the female Rhodnius prolixus, a blood-gorging triatomine and the vector of Chagas disease, we have identified the ecdysone response genes in the ovary using transcriptomic data. We then quantified the expression of the ecdysone response gene transcripts (E75, E74, BR-C, HR3, HR4, and FTZ-F1) in several tissues, including the ovary, following a blood meal. These results confirm the presence of these transcripts in several tissues in R. prolixus and show that the ecdysone response genes in the ovary are mostly upregulated during the first three days post blood meal (PBM). Knockdown of E75, E74, or FTZ-F1 transcripts using RNA interference (RNAi) was used to understand the role of the ecdysone response genes in vitellogenesis and egg production. Knockdown significantly decreases the expression of the transcripts for the ecdysone receptor and Halloween genes in the fat body and the ovaries and reduces the titer of ecdysteroid in the hemolymph. Knockdown of each of these transcription factors typically alters the expression of the other transcription factors. Knockdown also significantly decreases the expression of vitellogenin transcripts, Vg1 and Vg2, in the fat body and ovaries and reduces the number of eggs produced and laid. Some of the laid eggs have an irregular shape and smaller volume, and their hatching rate is decreased. Knockdown also influences the expression of the chorion gene transcripts Rp30 and Rp45. The overall effect of knockdown is a decrease in number of eggs produced and a severe reduction in number of eggs laid and their hatching rate. Clearly, ecdysteroids and ecdysone response genes play a significant role in reproduction in R. prolixus.


Introduction
Ecdysteroids play a crucial role in insect development, metamorphosis, and reproduction [1,2] Ecdysteroids include ecdysone (E), produced from cholesterol, and 20-hydroxyecdysone (20E), the biologically active hormone, produced from the conversion of E by cytochrome P450 enzymes. 20E binds to a heterodimeric nuclear hormone receptor consisting of the ecdysone receptor (homolog of the mammalian farnesoid X receptor) and utraspiracle (homolog of the mammalian RXR) (EcR /USP). The ecdysone receptor complex acts as a transcription aegypti have only three, A, B and C, and in the hemimetabolous species Blatella germanica, five isoforms are present [11,[39][40][41]. Additionally, E74, HR3, and BR-C have different mRNA isoforms and different expression patterns in insects; for example, there are two isoforms of the E74 gene in D. melanogaster and A. aegypti, and three isoforms of HR3 in B. germanica, named HR3-A, HR3-B1 and HR3-B2 [21]. Also, in D. melanogaster, several BRC isoforms are produced by alternative splicing among duplicated exons [9,17]. While there are two isoforms of FTZ-F1 in D. melanogaster, which are produced by alternative transcription and splicing [25,42], some insect species have only documented one transcript of FTZ-F1 [43,44], including R. prolixus [38]. The role of the individual participants in the hierarchy of ecdysteroid signaling, however, is still largely unknown during reproduction in female R. prolixus. In this paper, we identify and characterize the ecdysone response genes E75, E74, BR-C, HR3, HR4, and FTZ-F1 in R. prolixus and demonstrate that these genes are essential for normal egg production. We hypothesized that ecdysone response genes are required in 20E biosynthesis and oocyte maturation in the female ovary of R. prolixus.

Insects
R. prolixus were taken from a colony kept at 25˚C and~50% relative humidity and fed on defibrinated rabbit blood (Cedarlane Laboratories Inc., Burlington, ON, Canada). The experimental adult insects were previously blood fed as 5 th instars. Newly ecdysed adult females and males were separated and maintained at 28˚C with a 12 h light:12 h dark regime. Some experiments used mated adult females which had been fed at 10 days post ecdysis (PE) and only insects feeding 2.5-3 times their body weight was used.

Identification of gene sequences in the ovary transcriptome
An analysis of VectorBase was conducted to extract the sequences of the R. prolixus ecdysone response genes (E75, E74, BR-C, HR3, HR4, and FTZ-F1). ExPASy2 tool (www.expasy.org) was used to deduce amino acid sequences and a tBLASTn web portal (https://blast.ncbi.nlm. nih.gov/Blast.cgi) was used to run BLAST against the VectorBase genes. The high-level contigs matching with NCBI were confirmed by the e-value and amino acid sequence identity of the top contig hits. The ecdysone response genes were extracted from VectorBase with E74, E75, BR-C, and HR4 having one contig each. There are two contigs for FTZ-F1 (RPRC001915 and RPRC002968) with the top hit in BLAST for both being FTZ-F1β. Using Conserved Domain Database (CDD) software (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to analyze the main domain, it was found that RPRC002968 has both domains, the ligand-binding domain (LBD) and DNA-binding domain (DBD), while RPRC001915 is fragmented and has only the LBD domain. We therefore used RPRC002968 in this study. HR3 has two contigs, RPRC003681 and RPRC000824. While RPRC003681 has a complete DBD with N-terminus, it lacks the LBD while another contig RPRC000824 has only an LBD domain, suggesting that these are fragments of the same gene. The CDD analysis showed that E74 has an ETS with an N-and C-terminal. In addition, E75 and HR4 have DBD and LBD with an N-and C-terminal. BR-C sequences have complete main domain BTB (Broad-Complex, Tramtrack and Bric à brac). After confirming the extracted contig of the genes, their transcript expression in an ovary transcriptome was verified [45]. Phylogenetic trees were generated based on the sequences of the R. prolixus genes and other arthropod sequences using the MEGA Software (https://www.megasoftware.net). Evolutionary history was estimated using a maximum likelihood and a matrix-based model based on JTT [46]. Based on a matrix of pairwise distances estimated using the JTT model, the Neighbor-Joining and BioNJ algorithms were applied to an initial tree and the topology with the highest log likelihood value was selected. Thirty protein sequences were analyzed. The evolutionary analyses were conducted in MEGAX [47].

RNA extraction and reverse transcription/quantitative PCR (RT-qPCR)
Adult female R. prolixus tissues were dissected in cold autoclaved phosphate-buffered saline (PBS, 8.2 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 150 mM NaCl, 2.7 mM KCl). Total RNA was extracted according to the manufacturer's instructions using the TRIzol reagent (Invitrogen by ThermoFisher Scientific, MA, USA), and the final concentration and A260/280 ratio of purified RNA determined using a spectrophotometer DS-11+ (DeNovix Inc., Wilmington, DE, USA). To treat total RNA extracts with DNase, a DNase I (RNase-free) Kit (ThermoFisher Scientific Inc., Mississauga, ON, Canada) was used, according to the manufacturer's instructions. Reverse transcriptase (High-Capacity cDNA Reverse Transcription Kit, Applied-Biosystems, by Fisher Scientific, Mississauga, ON, Canada) was used to synthesize cDNA from 1 μg of total RNA. Random primers and 50 units of MultiScribe MuLV reverse transcriptase were used in accordance with the manufacturer's instructions. Quantitative PCR (qPCR) was performed as previously described [37], using 4 pmol of forward and reverse primers added to a Supergreen Low Rox Reagent (Wisent Bioproducts Inc, Saint-Jean-Baptiste, QC, Canada) and Advanced Master Mix (S1 Table). Although in R. prolixus only one ortholog for each of the ecdysone response genes has been reported, we cannot rule out isoforms, and so this presents a limitation on whether the qPCR is detecting only one isoform or different isoforms. The target gene expression was normalized using actin, Rp49 (60S ribosomal protein L32) or 18S ribosomal RNA (18S rRNA) as housekeeping genes. The expression of mRNA was calculated relative to 1000 copies of the average of the reference genes with the 2 −ΔCt method, while fold change was quantified relative to the expression of control samples, using the 2 −ΔΔCt method and the geometric mean of the reference genes [48,49].

Ecdysteroid ELISA
The 20E titer was measured in fed female adult insects at 4 and 5 days PBM. Hemolymph was collected using a Hamilton syringe (Hamilton Company, Reno, NV, USA), mixed with 100% methanol at a ratio of 1:3, and stored at -20˚C. The hemolymph 20E titers were quantified by competitive ELISA [37,50].

Double-stranded RNA design and synthesis
The E75, E74, and FTZ-F1 transcripts were downregulated using double-stranded RNAs (dsRNA). For E75, dsRNA primers cover LBD (conserved domain) and part of the C terminal, a less conserved region; for E74, primers cover part of the unique N-terminal region and the main conserved domain ETS domain; and for FTZ-F1, primers cover the N-terminal A/B domains. In general, 20E-responsive genes isoforms differ in the N-terminal. Using T7 RNA polymerase, PCR was used to synthesize non-overlapping fragments of the target gene. The dsRNA sequences were compared with the R. prolixus genome in VectorBase using BLAST. One high confidence hit was found for each dsRNA confirming their specificity. dsRNA was synthesized using the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA). dsARG (Ampicillin Resistance Gene) from the pGEM-T Easy Vector system (Promega, Madison, WI, USA) was used as control.
Significant knockdown of the transcripts was observed at these doses and days (S1 Fig). Following a blood meal on day 10 PE, all females were kept in cubicles with two males at 28˚C and 50% humidity. The cubicles were examined for the ejected spermatophores to verify mating. On day 4 PBM, the expression of the E75, E74, and FTZ-F1 transcripts in an individual ovary or fat body was determined as described above. RT-qPCR was also used to determine the levels of vitellogenin transcripts, Vg1 and Vg2, in the ovaries and fat body. A minimum of four independent biological samples were analyzed at each time point post injection. The ovaries of dsRNA-injected female R. prolixus were dissected and photographed at 4 days PBM using a Leica DVM6 digital microscope (Leica Microsystems, Wetzlar, Germany).

Egg-laying assay
Mated and fed females begin to lay eggs 5-6 days after a blood meal, and egg-laying typically occurs over 20-30 days post feeding [45]. The eggs laid by dsRNA-injected insects (as described above) were counted for 15 days PBM. Eggs were photographed using a Leica DVM6 digital microscope (Leica Microsystems, Wetzlar, Germany). To measure the volume of the eggs, 15 eggs were randomly selected from each dsRNA group and volume was calculated using the equation 1/6 π(length)(width) 2 [51]. The hatching rate was also determined.

Statistical analyses
Results are expressed as mean ± SEM. Graphs were created using GraphPad Prism 7 (Graph-Pad Software, San Diego, California, USA). All data sets passed the normality and homoscedasticity tests. For experiments with multiple groups, one-way ANOVA and Tukey's post-hoc test was used, while Student's t-tests were used for comparison between two values. The statistical significance of each case was determined by a P value of < 0.05.

Identification of ecdysone response genes in the ovary transcriptome of R. prolixus
Ecdysone response genes E75, E74, BR-C, HR3, HR4, and FTZ-F1 were identified using insect orthologs in the GenBank database. The R. prolixus sequences were verified by BLAST against the GenBank database. The R. prolixus genes are all full-length except for HR3 and HR4, which is a fragment (S2 Table). MEGAX software was used to generate a phylogenetic tree. The phylogenetic tree illustrates that the ecdysone response genes of R. prolixus cluster with their orthologous sequences from other arthropod species (Fig 1). All of the ecdysone response genes are expressed in the ovary transcriptome (S2 Table).

Tissue transcript expression of ecdysone response genes
Transcript levels of the ecdysone response genes (E75, E74, BR-C, HR3, HR4, and FTZ-F1) were quantified from adult female tissues at 10 d PE from unfed insects using qPCR. Eight tissues were examined: central nervous system (CNS), anterior midgut (AMG), Malpighian tubules (MTs), hindgut (HG), fat body (FB), ovaries (OV), and oviduct (Ovid) + spermatheca (Sp). Transcript expression varies among the tissues collected (Fig 2A-2F), but overall, E75 is the most highly expressed, and HR3 is the lowest expressed transcript. With regard to the reproductive tissue, E75, E74, HR3, HR4, and FTZ-F1 transcripts are all expressed in the ovaries, but BR-C mRNA transcript expression is relatively low (Fig 2A-2F). In addition, E75, E74, HR4, and FTZ-F1 transcripts are expressed in the oviducts and spermatheca, along with much lower levels of BR-C and HR3 transcripts. In other tissues, E75, E74, BR-C and FTZ-F1 (but not HR3 and HR4) have high levels of expression in the CNS and hindgut. E75 and E74 are also highly expressed in the fat body. There is a lower expression for HR3, HR4, and FTZ-F1 in the anterior midgut and Malpighian tubules (Fig 2A-2F).

The effects of a blood meal on transcript expression levels of the ecdysone response genes in the ovary
The transcript expression of ecdysone response genes was quantified in the ovaries of unfed adult females at 10 d PE, and then at days PBM (Fig 3A-3F). All ecdysone response genes are elevated in the first few days following a blood meal. E75 transcript levels are elevated between days 1 and 6 PBM, while E74, BR-C and FTZ-F1 levels are elevated in the second and third days PBM, but then decrease over the following days. There is an elevation in HR3 on day 2, 3 and 4 PBM, followed by a decrease on days 5-6 PBM. HR4 transcript levels are elevated by day 2 PBM, then decrease on the following days PBM (Fig 3A-3F).

The effects of knockdown of E75, E74 or FTZ-F1 transcripts on transcript expression of the Halloween genes, ecdysone receptor, and ecdysone response genes in the ovary, and on hemolymph ecdysteroid titer
In response to the knockdown of E75, the expression of all Halloween genes (spook, phantom, disembodied, shadow, and shade) in the ovary decreases (Fig 4A-4E). While knockdown of E74 downregulates transcripts for spook and phantom in the ovary, the transcripts for shadow, disembodied, and shade are not affected (Fig 4F-4J). dsFTZ-F1 injection downregulates phantom, disembodied, and shadow transcripts in the ovary, increases spook expression, but does not affect shade (Fig 4K-4O).
Knockdown of E75 or E74 expression downregulates the expression of the EcR and USP transcript expression in the ovary; however knockdown of FTZ-F1 downregulates EcR expression but upregulates USP (Fig 5A-5F).
The knockdown of the ecdysone response genes, E75, E74, or FTZ-F1, alters expression of other ecdysone response genes with dsE75 significantly reducing expression of E74 and HR3 in the ovary, but not the transcripts for BR-C and FTZ-F1 (Fig 6A-6D). Similarly, dsE74 reduces the level of expression of E75 and HR3 transcripts in the ovary but does not alter expression of BR-C or FTZ-F1 (Fig 6E-6H). Knockdown with dsFTZ-F1 results in a downregulation of the expression of E75, BR-C and HR3 but does not alter E74 expression in the ovary (Fig 6I-6L).
Knockdown of transcripts for E75and FTZ-F1 reduces hemolymph ecdysteroid levels significantly compared with control insects on days 4 and 5 PBM (Fig 7A and 7B).

Effects of knockdown of ecdysone response genes on egg production in adult females
Unfed adult females 7 d PE were injected with dsRNA and then blood fed and mated on 10 d PE. The efficacy of dsRNA treatment was monitored in the ovaries and fat body 4 d PBM (during vitellogenesis) and E75, E74, and FTZ-F1 transcript levels found to be significantly reduced in knockdown insects (S1 Fig). At 4 d PBM, Vg1 and Vg2 transcript expression levels are significantly lower in the fat body and ovaries following dsE75, dsE74, or dsFTZ-F1-injection as compared to dsARG-injection (Figs 8A-8D, 9A-9D and 10A-10D). At day 4 PBM, the ovaries of female R. prolixus injected with dsRNA were dissected and photographed. E75, E74, or FTZ-F1 knockdown significantly reduces oogenesis, with ovaries producing fewer mature oocytes when compared to dsARG-injected insects. The number of growing follicles (vitellogenic follicles) is also significantly reduced in dsE75, dsE74, or dsFTZ-F1-injected insects compared to dsARG treatment (Figs 8E, 8F, 9E, 9F, 10E and 10F). Knockdown of E75, E74, or FTZ-F1 also dramatically decreases the number of eggs laid per female (to 24%, 36%, and 20%, respectively) compared to controls and decreases the hatching rate (to 52%, 79.5%, 90%, respectively) of those few eggs laid (Figs 8G, 9G and 10G). The egg volume of those eggs laid

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from dsE74 or dsFTZ-F1-injected insects is smaller, compared to those from controls, while dsE75 injection does not alter egg volume (Figs 8J, 9J and 10J). Moreover, both dsE75 and dsE74-injected insects produce some eggs that have an abnormal egg shape (lateral compression) (Figs 8K and 9K) compared with the normal shape (Figs 8J and 9J). The eggs produced by dsFTZ-F1-injected insects appear normal in shape (Fig 10J).
The effect of knockdown of E75, E74, or FTZ-F1 on choriogenesis was studied by quantifying the transcript expression for the choriogenesis genes, Rp30 and Rp45, in ovaries on day 4 PBM. Interestingly, Rp30 and Rp45 transcripts are significantly reduced in insects injected with dsE75, dsE74, or dsFTZ-F1 (Fig 11).

Discussion
Insect development, molting, and metamorphosis are dependent on 20E. Cytochrome P450 monooxygenases, encoded by Halloween genes spook (spo), phantom (phm), disembodied (dib), shadow (sad), and shade (shd), are involved in ecdysteroid biosynthesis. The overall ecdysteroid signal is regulated by a large number of transcription factors (ecdysone response genes) [8]. In terms of ecdysteroid biosynthesis, transcription factors have mainly been studied in relation to their function in prothoracic glands (PGs) in non-adult insects. However, ecdysteroid biosynthesis occurs in adult stages, in the absence of PGs, with the ovaries as the site of

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Ecdysteroid signaling and egg production in Rhodnius prolixus. biosynthesis [6,52]. Furthermore, and importantly, these transcription factors are highly expressed in non-ecdysteroidogenic tissues since they participate in other ecdysteroid-mediated effects. Our previous study illustrated the importance of ecdysteroid for egg production in R. prolixus, and knocking down the ecdysone receptor and shade reduces the number of eggs made and laid, and influences the maturation of oocytes, suggesting ecdysteroid influences vitellogenesis. Furthermore, the abnormal shape of the few eggs laid by insects injected with dsEcR, dsUSP, or dsshade, as well as the decrease in hatching of those eggs, suggests that the eggs are not capable of successful choriogenesis. This was supported by the significant reduction in expression of the chorion gene transcripts (Rp30 and Rp45) in insects injected with dsEcR, dsUSP, or dsshade [37]. In the current study, we have investigated the role of the ecdysone response genes in the regulation of egg production by first identifying them in an ovary transcriptome, examining their tissue distribution, and then examining the effects of knockdown of their transcript expression. Here we find that the ecdysone response genes participate in the 20E regulatory hierarchy controlling vitellogenesis and chorion formation in female R.

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Ecdysteroid signaling and egg production in Rhodnius prolixus.

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Ecdysteroid signaling and egg production in Rhodnius prolixus.
prolixus, and knockdown of their transcripts disrupts egg production. We identified E74, E75, HR3, HR4, and FTZ-F1 with significant expression in the ovary, fat body, and brain, which is consistent with reports of other insects [8,28,[53][54][55][56][57]. The expression of ecdysone response genes in the ovary increases following a blood meal in R. prolixus, and likely mediates feedback to increase ecdysteroid biosynthesis via the Halloween genes, and thereby increase hemolymph Females were injected with dsRNA and blood fed as described in Materials and Methods. Relative transcript levels were measured using RT-qPCR and analyzed using the 2 −ΔΔCt method. Data are means ± SEM (n = 4-7). Rp49 and β-actin were used as reference genes. Statistical analysis was performed by Student's t-test. � p < 0.05, �� p < 0.01, ���� p<0.0001, ns = not significant. https://doi.org/10.1371/journal.pone.0283286.g006

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ecdysteroid titer as is necessary during vitellogenesis for egg production. Knockdown of the ecdysone response genes in R. prolixus by RNAi significantly decreases Vg1 and Vg2 expression in the fat body and ovaries, reduces the titer of ecdysteroid in the hemolymph, and interferes with normal egg production. Further evidence of feedback is shown by knockdown of E75, E74 or FTZ-F1, which downregulates the transcripts in the fat body and ovary for EcR and USP at day 4 PBM. In addition, knockdown of E75, E74 or FTZ-F1 expression results in a

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downregulation of all Halloween gene transcripts at day 4 PBM (spook, phantom, disembodied, shadow, and shade). Moreover, dsE75, dsE74, or dsFTZ-F1 mostly reduces the expression of ecdysone response genes in the ovary and fat body. These data suggest ecdysone response genes increase the expression of steroidogenic enzymes in the ovary and fat body via a positive feedback loop, resulting in dramatic increases in ecdysteroid levels in the hemolymph. This data confirms the complex interaction between ecdysteroid biosynthesis, its receptor and the ecdysone response genes.
Ecdysone response genes have previously been reported to be induced by ecdysteroids and are implicated in the regulation of vitellogenesis and female reproduction in many insects [7,56,58]. In A. aegypti the blood meal triggers the 20E cascade with a high expression of genes in the ovary and fat body leading to activation of yolk protein precursor genes in the fat body [30,59]. Moreover, in the fat body, EcREs in the 5-upstream region of the Vg gene have been identified, along with those of E74, E75, and BR-C, indicating direct and indirect regulation by 20E [30,31,60]. In addition, in A. aegypti, a blood meal also activates three early E75 isoforms and elevates their expression in the fat body during vitellogenesis, with two peaks appearing at 4 h and 24 h PBM [59]. Moreover, a blood meal induces an upregulation of AaE74 transcript levels in the fat body and ovary, with the highest levels occurring during the peak of vitellogenesis [61]. Our data supports the presence of the ecdysteroidogenic transcription factors in the fat body and ovary and also their upregulation following a blood meal. . dsE75-injection affects ovarian phenotype in comparison to dsARG-injected controls (E and F) (n = 4). Eggs laid per female over 15 days PBM (G) (n = 15) and egg volume (H) (n = 15). Phenotypes of laid eggs from insects injected with dsARG (I) or injected with dsE75 (J and K) (n = 15). At 4 days PBM, ovaries were dissected, and images were taken. Rp49 and β-actin were used as reference genes. The transcript levels were quantified using RT-qPCR and analyzed using the 2 −ΔΔCt method. Results are presented as mean ± SEM. Student's t-test was used for statistical analysis. � p < 0.05; �� p < 0.01, ���� p < 0. 0001, ns = not significant. https://doi.org/10.1371/journal.pone.0283286.g008

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Disruption of FTZ-F1 expression in D. melanogaster and T. castaneum causes abnormal follicle development, a decrease in the synthesis of Vg, blocked maturation of the oocyte and ovulation failure [24,62]. In addition, in N. lugens, Vg expression is significantly reduced 24 hours following injection of dsE74A and the decline is more pronounced 48 hours after injection [63]. Knockdown of E75, E74, or FTZ-F1 in R. prolixus decreases egg production, resulting in fewer eggs being laid than controls, and a reduced hatching rate, especially for knockdown of the transcript for E75. A small egg size was found from the dsE74 and dsFTZ-F1-injected insects, but interestingly, this did not appear to affect the hatching rate of eggs laid by dsFTZ-F1-injected insects. In part these results could be caused by lower ecdysteroid titer in the hemolymph coupled with lower expression of vitellogenin genes, influencing the maturation of oocytes and their growth. We have previously reported that in R. prolixus, disruption of ecdysteroid signaling, via knockdown of ECR, USP and Halloween genes, affects vitellogenin expression, chorion gene expression, oocyte maturation, and egg-laying [37], and the present data illustrates that the effects of ecdysteroids are mediated by the ecdysone response genes. In T. castaneum, egg-laying is completely blocked with the knockdown of E75, HR3, EcR, USP, SVP, FTZ-F1 and HR4 [24]. Moreover, knockdown of ECR and BR-C in Cimex lectularius results in a reduction in the number of eggs laid [64]. Also, in N. lugens, silencing E93, one of the early ecdysone response genes, suppresses ovary development, resulting in fewer eggs produced [65]. In addition to playing a role in egg production, ecdysteroid signaling is involved in

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choriogenesis in adult female desert locusts through the nuclear receptor complex [65,66]. The ecdysone receptor and shade play important roles in the regulation of vitellogenesis in R. prolixus [37]. 20E induces Vg synthesis in the fat body, but how it regulates vitellogenesis is unclear. Currently, we do not know whether 20E influences oocyte maturation directly, or whether the signals released by maturing oocytes influence vitellogenesis. This study investigated the role of genes downstream of 20E signaling on egg production and transcription of vitellogenin. As a result, RNAi knockdown of genes that encode proteins involved in ecdysteroid signaling result in severe defects in the maturation of the oocytes and egg production. Our findings are consistent with reports showing that the downregulation of E75, E74, HR3, and FTZ-F1 affects the expression of Halloween genes and other transcription factors in the ovary and fat body. Several transcription factors have been identified as responsible for Halloween gene expression. In the PGs, E75, FTZ-F1, and HR3, which are downstream of 20E-EcR signaling, regulate ecdysteroid biosynthesis [8,67]. In D. melanogaster, knockdown of BR-C and HR3 in the ring gland leads to a reduced expression of ecdysone biosynthesis related genes (phantom, disembodied and shadow) while downregulation of E75 reduces the expression of phantom [7,8,11,[67][68][69][70]. In R. prolixus females E75 expression is also influenced by dsFTZ-F1and dsE74, whereas HR3 expression is influenced by dsE75, dsE74 and dsFTZ-F1. Similar feedback of the ecdysone response genes on genes responsible for ecdysteroid biosynthesis or the 20E-EcR pathway have been shown in other studies. For example, the levels of dsFTZ-F1-injection affects ovarian phenotype in comparison to dsARG-injected insects (control) (E and F) (n = 4). Eggs laid per female over 15 days PBM (G) (n = 15) and egg volume (H) (n = 15). Phenotypes of laid eggs from insects injected with dsARG (I) and insects injected with dsFTZ-F1 (J-K) (n = 15). At 4 days PBM, ovaries were dissected, and images were taken. Rp49 and β-actin were used as reference genes. The transcript levels were quantified using RT-qPCR and analyzed using the 2 −ΔΔCt method. Results are presented as mean ± SEM. Student's t-test was used for statistical analysis. ��� p < 0.001, ���� p < 0. 0001. https://doi.org/10.1371/journal.pone.0283286.g010

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mRNA of EcR and USP are decreased by 80-90% in B. mori E93 RNAi larvae compared to their control counterparts [71]. Furthermore, in B. mori E75 RNAi disrupts the 20E-triggered transcriptional cascade by lowering EcR-B1 and USP protein levels [72] and in D.

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melanogaster early L3 larvae, RNAi of FTZ-F1 decreases EcR expression, indicating that FTZ-F1 is necessary to maintain EcR expression in this stage [69].
This study helps us understand how ecdysone response genes regulate Vg expression in R. prolixus and the feedback between ecdysteroid and ecdysone response genes. However, how these transcription factors regulate Halloween gene transcription still needs further investigation.
Supporting information S1 Fig. Efficiency of knockdown of E75, E74 and FTZ-F1  Knockdown of these genes in the FB downregulates the transcript levels of some of the ecdysone response genes in the fat body at 4 days post blood meal of adult female R. prolixus. Females were injected as described in Materials and Methods. Relative transcript levels were measured using RT-qPCR and analyzed using the 2 −ΔΔCt method. .Rp49 and β-actin were used as reference genes. Data indicate means ± SEM (n = 4-6). � p < 0.05, �� p < 0.01, ��� p<0.001, ���� p < 0. 0001; ns = not significant. Statistical analysis was performed by Student's t-test. (DOCX) S1  genes (E75, E74, BR-C, HR3, HR4 and FTZ-F1) sequences from the R. prolixus genome and ovary transcriptome. (DOCX)